There is a continuing need in medical practice and research, and in analytical and diagnostic procedures for rapid and accurate determinations of chemical and biological substances which are present in various fluids, such as biological fluids. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, metabolites, toxins, viruses, microorganisms or nucleic acids in human or animal body fluids or tissues must be determined rapidly and accurately for effective research, diagnosis or treatment.
A wide variety of analytical methods have been developed to detect the substances noted above. Generally, the state of the art has advanced to such a degree that analytical and diagnostic methods have become highly reliable and suitable for automation or for use with test kits which can be readily used in doctors' offices or at home. Most of such methods rely on what are known in the art as "specific binding" reactions in which an unknown substance to be detected (known as a "ligand") reacts specifically and preferentially with a corresponding "receptor" molecule. Most well known specific binding reactions occur between immunoreactants, such as antibodies and antigens (foreign substances which produce immunological responses), but other specific binding reactions (such as between avidin and biotin and a sugar with a lectin) are well known.
Methods in the art using specific binding reactions generally require that one or more or both of the reactants be immobilized on a solid substrate of some type, so that unreacted (and generally water-soluble) materials can then be separated from the water-insoluble reaction product (often called a "complex"). In addition, such immobilized reactants can be used in affinity chromatography to remove a desired biologically active material from a mixture of such materials.
U.S. Pat. No. 4,278,651 (issued Jul. 14, 1981 to Hales) relates to a supported receptor for use in an assay for a ligand in which the solid support contains a water insoluble polymer having available at least one active functional group which is either carboxyl, isothiocyanate, N-hydroxysuccinimid, imidazolide, bromoacetyl, maleimide or diazomethylene. The receptor having been covalently linked to the support through the active functional group. Generally, the support is a large core-shell particle having an outer porous coating as the shell which also has the necessary functional groups. The core of the particle provides structural integrity for the porous shell materials.
It would be expected that porous particles would provide greater surface area over non-porous particles. This, in practice, is not the case. Depending on the size of the pores, porous particles are relatively inefficient for use with large molecules of biological interest. For example, a useful ligand may be attached to a porous bead in one of the pores. The biological species which it is desired to eliminate or separate from the liquid stream may be so large as not to be able to get down into the pores and reach the ligand. Conversely, immobilization of a large affinity ligand can only take advantage of a small portion of the total surface area since the ligand itself cannot penetrate the porous well. Thus, the efficiency of binding is diminished and the apparent surface area advantage of such porous particles becomes illusory.
Acrylic acid-based photopolymerizable compositions have been prepared which are capable of binding bioactive substances after being photopolymerized, as described in U.S. Pat. No. 4,451,569 (issued May 29, 1984 to Schneider et al). These compositions may be applied as a coating on a carrier substrate, photopolymerized and a bioactive substance fixed thereto. The composition contains acrylic acid, a photoinitiator, a photopolymerization activator and adhesion promoter, and a copolymerizable olefinic monomer which contains a active functional group capable of binding bioactive substances. The olefinic monomer is preferably N-hydroxysuccinimidacrylate, N-hydroxysuccinimid amidocaproate, epoxypropyl acrylate or 2-isocyanato-ethyl acrylate.
Also, biologically active substances have thus been immobilized to advantage on particulate substrates such as polymeric particles, animal and human erythrocytes, bacterial cells and other materials known in the art. In some cases, the particulate substrates are fashioned or chemically treated to provide active groups on their outer surfaces for appropriate reaction with the biological substance. If the particulate substrate is a polymeric material, it often can be prepared from monomers having the appropriate active groups.
For example, carboxylated latex particles have been used to prepare diagnostic reagents, as noted in U.S. Pat. No. 4,181,636 (issued Jan. 1, 1980 to Fischer). The described particles are prepared using a carboxyl-containing monomer such as acrylic acid, methacrylic acid, itaconic acid, aconitic acid, fumaric acid or maleic acid. Similar particles are described in U.S. Pat. No. 3,857,931 (issued Dec. 31, 1974 to Hager), U.S. Pat. No. 4,138,383 (issued Feb. 6, 1979 to Rembaum et al) and U.S. Pat. No. 4,264,766 (issued Apr. 28, 1981 to Fischer).
For example. U.S. Pat. No. 4,710,525 (issued Dec. 1, 1987 to Kraemer) relates to certain polymer particles dispersible to form a latex, to latices of such polymer particles, and to methods for immobilizing (i.e., bonding or fixing) a biologically active substance on such particles. These particles have a core-shell construction and comprise groups in the shell region which are suitable for covalent fixation thereto of a biologically active substance. The shell construction is also hydrophilic and crosslinked. The crosslinking is necessary to prevent dissolution of the very hydrophilic shell polymer. The crosslinked hydrophilic matrix of Kraemer has the disadvantage of being nonabsorptive towards antibodies and other proteins to be adsorbed or immobilized on the surface of the antibody or protein. A material that adsorbs to a hydrophilic surface can be more easily displaced than if it were adsorbed to a more hydrophobic surface.
Two known monomers, N-acryloyloxysuccinimid and N-(6-methacrylamidohexanoyloxy)succinimid, have been polymerized to form polymers. These monomers are generally water-insoluble, but are difficult to copolymerize with oleophilic monomers by emulsion polymerization in water and are not readily polymerized to form monodisperse particles.
Notwithstanding the current status of the arts of medical practice and analytical and diagnostic procedures, there is a need in the industry for a method of preparing biologically active reagents having water-insoluble, non-porous particles of a non-crosslinked copolymer useful in this invention.